Cytokine 73 (2015) 36–43
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Pro-inflammatory effects of interleukin-35 in rheumatoid arthritis Mária Filková a,⇑, Zdenka Vernerová b, Hana Hulejová a, Klára Prajzlerová a, David Veigl c, Karel Pavelka a,d, Jirˇí Vencovsky´ a,d, Ladislav Šenolt a,d a
Institute of Rheumatology, Prague, Czech Republic Institute of Pathology of the 3rd Faculty of Medicine, Charles University in Prague, Prague, Czech Republic c 1st Orthopedic Clinic, 1st Faculty of Medicine and Faculty Hospital Motol, Charles University in Prague, Prague, Czech Republic d Department of Rheumatology, 1st Faculty of Medicine, Charles University in Prague, Czech Republic b
a r t i c l e
i n f o
Article history: Received 16 March 2014 Received in revised form 30 October 2014 Accepted 23 January 2015
Keywords: Rheumatoid arthritis Synovial tissue Inflammation Interleukin-35
a b s t r a c t Objective: Interleukin-35 (IL-35) is a heterodimeric member of the IL-12 family consisting of p35/IL-12a and EBI3/IL-27b subunits. Expressed in murine Treg cells, IL-35 controls inflammatory diseases in mouse models. However, human IL-35 is expressed in Teff cells rather than in Treg cells and is shown to be upregulated under inflammatory conditions. Our aim was to examine the involvement of IL-35 in the pathogenesis of rheumatoid arthritis (RA). Methods: Immunohistochemical and immunofluorescence analysis was used to determine the expression and localization of IL-35 and its subunits (p35/EBI3) and IL-35 receptor (IL12Rb2/gp130) in RA, osteoarthritis (OA) and psoriatic arthritis (PsA) synovial tissues. Expression of p35/EBI3 subunits and release of inflammatory cytokines upon stimulation with IL-35 were assessed in RA synovial fibroblasts (SFs) and peripheral blood mononuclear cells (PBMCs). Results: Both IL-35 and its subunits were upregulated in RA in comparison with OA or PsA synovium. Using cell-specific markers, p35 and EBI3 were identified in macrophages, dendritic cells, SFs, and T as well as B cells in RA synovium. Both p35 and EBI3 were induced by TNFa in RASFs and PBMCs. IL-35 dose-dependently upregulated release of pro-inflammatory mediators IL-1b, IL-6 and MCP-1 in PBMCs. While gp130 receptor subunit was upregulated in RA synovium and was expressed in RASFs and PBMCs, there was no difference in IL12Rb2 expression subunit among tissues and its presence in RASFs was lacking. Conclusion: Upregulation of IL-35 at sites of inflammation in RA and its pro-inflammatory potential suggests that IL-35 might play an important role in RA pathogenesis. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation and hyperplasia of the synovial tissue, increased production of inflammatory mediators, and subsequent joint destruction. It is now well established that macrophages, activated T and B cells, and their products including tumor necrosis factor-a (TNFa) and interleukin (IL)-1b and IL-6 play a significant role in the pathogenesis of RA [1]. Although currently available biologic treatment targets these specific cellular and molecular mechanisms, only a minority of patients achieve remission [2]. Therefore, studies of various molecular pathways have focused on targets for future therapies [3].
⇑ Corresponding author at: Institute of Rheumatology, Na Slupi 4, Prague 128 50, Czech Republic. Tel.: +420 234 075 323; fax: +420 224 914 451. E-mail address: [email protected] (M. Filková). http://dx.doi.org/10.1016/j.cyto.2015.01.019 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
IL-35 is a newly described cytokine that belongs to the IL-12 family consisting of the heterodimeric cytokines IL-12, IL-23, IL-27, and IL-35 [4]. Similar to other IL-12 members, IL-35 consists of a chain (p35/IL-12a) that can also dimerize with p40 to form IL-12, and b chain (EBI3/IL-27b) sharing a partnership with the p28 subunit of IL-27. Like the cytokine chains, also subunits of IL-35 receptor are shared among IL-12 family members. IL-35 receptor consists of IL12Rb2 (IL-12 receptor component) and gp130 (IL-27 receptor component) activating subsequently STAT1 and STAT4 signaling pathways [5]. While p35 is ubiquitously expressed, EBI3 (Epstein–Barr virusinduced gene 3) is selectively produced and is highly inducible [6,7]. Although the association between p35 and EBI3 was described several years ago, the biologic role of this heterodimer has remained undetermined until now [8]. However, data on animal models remain controversial. While some support anti-inflammatory activities of IL-35 in inflammatory bowel disease, collagen induced arthritis (CIA) or other autoimmune conditions given its
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effect on Treg, Breg and associated cytokines [9–13], other data are suggestive of its pro-inflammatory properties. IL-35 was shown to act as an inflammatory mediator in Lyme arthritis or CIA under different experimental settings [14,15]. Recently, IL-35 was shown to convert proliferative Foxp3-T cells into suppressive iTR35 cells that mediated their suppression exclusively via IL-35 and to stimulate a new Treg CD39+ subset capable of controlling activated lymphocytes in a CIA model [11,13]. There is evidence that EBI3 is a downstream target of Foxp3 in mouse Treg cells [9], however, induction of Foxp3 activity does not upregulate EBI3 or p35 in humans [16]. These data show that IL-35 is not exclusively an anti-inflammatory cytokine and its role in different inflammatory animal models or human Treg cells remains contradictive [7]. Therefore IL-35 may play different roles in mice and humans. These reports bring firm evidence that IL-35 plays a role in autoimmune inflammatory diseases. Therefore, the aim of this study was to analyze the expression of IL-35 and its subunits in synovial tissue of patients with RA, psoriatic arthritis (PsA) and osteoarthritis (OA), and to investigate possible involvement of IL35 in the pathogenesis of RA.
2. Methods 2.1. Immunohistochemistry Synovial tissue specimens were obtained during reconstructive hand surgery or wrist synovectomy in patients with RA (n = 5), during total knee joint prosthesis in patients with OA (n = 5) and PsA (n = 2). Patients with RA fulfilled the 1987 revised criteria of the American College of Rheumatology (ACR), patients with OA met the ACR criteria for OA of the knee and patients with PsA met CASPAR diagnostic criteria [17–19]. Written informed consent from each participant was obtained prior to enrollment, and the study was approved by the local ethics committee at the Institute of Rheumatology in Prague. Formaldehyde-fixed, paraffin-embedded sections (5 lm) were deparaffinized and rehydrated. Endogenous peroxidase activity was inhibited by 3% hydrogen peroxide in methanol for 30 min followed by 15 min of rinsing in tap water. Non-specific activity was avoided by pretreatment of sections with 1% normal bovine serum in phosphate-buffered saline (PBS) for 2 h. Immunohistochemical labeling was performed after antigen retrieval in Target retrieval solution pH 6.0 (Dako, Cytomation, Glostrup, Denmark) for 30 min at 98 °C and slides were incubated for 1 h at room temperature either with rabbit polyclonal anti-human CD130 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:2000, with rabbit polyclonal anti-human IL-12a antibody (Abcam, Cambridge, UK) diluted 1:4000, or with mouse monoclonal anti-human antibody to IL-35 (clone 15K8D10, Acris antibodies, San Diego, CA, USA) diluted 1:2000. All antibodies were diluted in the ChemMate antibody diluent (Dako, Cytomation, Glostrup, Denmark). Another immunohistochemical labeling was performed after antigen retrieval in Target retrieval solution pH 9.0 (Dako, Cytomation, Glostrup, Denmark), for 30 min at 98 °C and slides were incubated 1 hour at room temperature either with rabbit polyclonal anti-human IL12RB2 antibody (Bioss antibodies, Woburn, Massachusetts, USA) diluted 1:300 in ChemMate antibody diluent (Dako, Cytomation, Glostrup, Denmark), or rabbit polyclonal anti-human EBI3 antibody (Lifespan Biosciences, Seattle, WA, USA) diluted 1:100 in the same antibody diluent. The Envision kit (Dako, Cytomation) was used to visualize positive slides. The chromogen 3,3-diaminobenzidine (Liquid DAB + Substrate, Dako, Cytomation) was applied to all sections, and counterstaining was performed with Mayer’s hematoxylin. Isotype IgG (Dako, Cytomation) was used
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as a negative control. All slides were analyzed semi-quantitatively using a Nikon Eclipse E600 microscope (Nikon, Melville, NY, USA) in a blind fashion by two independent readers. The analysis included ten different fields throughout the biopsies separately in both lining and sublining layers (magnification 400), and a global score was assigned using a four-point scale: 0 represented negative staining intensity, and scores of 1–3 represented weak, moderate, and strong staining intensity, respectively. 2.2. Laser scanning confocal microscopy Confocal microscopy double immunolabeling was performed as follows: Slides were simultaneously incubated after antigen retrieval in 0.1 mol/l citrate buffer, pH 6.0, for 40 min with primary mouse anti-human CD20 monoclonal antibodies, clone L26, anti-human CD68 monoclonal antibodies, clone PG-M1, anti-human CD1a monoclonal antibodies, clone O1; anti-human CD3 monoclonal antibody, clone PC3; rabbit anti-human CD3 polyclonal antibodies or mouse anti-human vimentin monoclonal antibodies, clone 3B4 (all Dako, Cytomation), respectively. Further, we used primary rabbit anti-human EBI3 polyclonal antibodies (Lifespan Biosciences) or mouse anti-human IL-12a monoclonal antibodies (Santa Cruz, Santa Cruz, CA, USA). After 1 h, simultaneous incubation of the specimen was performed with FITC/CY5–conjugated secondary antibodiesAlexa Fluor 488 goat anti-mouse IgG (Invitrogen, Basel, Switzerland) and Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen) in the case of EBI3. In the case of IL-12a, we used FITC/CY5– conjugated secondary antibodies Alexa Fluor 488 goat anti-mouse IgG2 or IgG3 (Invitrogen) or Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 568 goat anti-mouse IgG1 (Invitrogen). All of these antibodies were diluted in PBS and all incubations took place at 20 °C for 30 min. The slides were mounted in Mowiol (Hoechst, Frankfurt am Main, Germany) and analyzed using a confocal laser scanning microscope, the Nikon Eclipse TE2000 (Nikon). Simultaneous excitation with anargon–krypton laser at wavelengths of 488 nm and 548 nm and appropriate 515 ± 15 and 590 ± 15 nm band-pass fluorescence filters were used for excitation and detection (in separate channel detection mode), respectively. 2.3. Cell cultures and stimulation assays Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll density gradient centrifugation from blood samples from healthy donors. Freshly isolated PBMCs were incubated in advanced RPMI medium 1640 (Invitrogen) supplemented with Lglutamine (Gibco, Carlsbad, CA, USA) and then stimulated for 6 or 24 h with the following agents: TNFa (10 ng/ml; R&D Systems, Abingdon, United Kingdom), IL-35:Fc human recombinant protein (25, 50, 100 ng/ml; ALEXIS Biochemicals, Enzo Life Sciences, Lausen, Switzerland), control: Fc fusion human recombinant protein (100 ng/ml; ALEXIS Biochemicals, Enzo Life Sciences), and polymyxin B sulfate (5 lg/ml; Sigma–Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 humidified atmosphere. RA synovial fibroblasts were isolated from synovial tissues as described previously [20] and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal calf serum (FCS) at 37 °C in a 5% CO2 humidified atmosphere. Synovial fibroblasts between passages 4 and 8 were stimulated with TNFa (10 ng/ml) or IL-35 (25, 50, 100 ng/ml) for 6 and 24 h. Cells were lysed in RLT buffer (QIAGEN, Hilden, Germany) after 6 and 24 h, and cell culture supernatants were collected after 24 h. Samples were stored at 80 °C until use. 2.4. RT-PCR analysis Total RNA was isolated using a MagNA Pure Compact RNA Isolation Kit for the MagNA Pure Compact Instrument (Roche, Mann-
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heim, Germany), and reverse transcription was performed with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). For real-time polymerase chain reaction (RT-PCR), TaqMan gene expression assays (Applied Biosystems) were used, and the reaction was performed with a 7900HT Fast Real-Time PCR System (Applied Biosystems). Data were analyzed using the dCt method for relative quantification with 18S as an endogenous control. 2.5. ELISA Cytokines IL-1b, IL-6, TNFa and monocyte chemoattractant protein-1 (MCP-1) released into the cell culture supernatants were measured by ELISA kit (RayBiotech, Norcross, GA,USA) and IL-8 was measured using IL-8 DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA). The absorbance was measured at 450 nm using an ELISA reader (Tecan Sunrise, Salzburg, Austria). 2.6. Statistical analysis The Mann–Whitney U-test, paired t-test, Kruskal–Wallis and Friedman test including Dunn’s multiple comparisons were used
where appropriate. To calculate the additive or synergistic effect of combined treatment of IL-35 plus TNFa on the expression of cytokines/chemokines, the observed (O; concentrations obtained for the combination of TNFa and IL-35) to expected (E; sum of the independent concentrations obtained for TNFa and IL-35, respectively) ratio (O:E) was calculated. The significance of differences between observed and expected concentrations was determined by paired t-tests. P values less than 0.05 were considered statistically significant. The analysis was performed and graphs created using GraphPad Prism 5 (version 5.02; GraphPad Software, La Jolla, CA, USA). 3. Results 3.1. IL-35 and its subunits (p35/EBI3) are upregulated in RA synovial tissue We found that the expression of both IL-35 subunits p35 and EBI3 in synovial tissue was present in patients with RA and that the staining intensity was significantly enhanced compared with patients with OA or PsA (Fig. 1). There was consistent expression of both IL-35 subunits within the mononuclear inflammatory
Fig. 1. Expression of p35 and EBI3 subunits of IL-35 in rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis (PsA) synovial tissues. Higher expression of p35 in RA (A) than in OA (B) and PsA (C) synovial tissue was demonstrated by semi-quantitative analysis (M). Similarly, overexpression of EBI3 in RA (E) compared with OA (F) and PsA (G) synovial tissue was revealed (N). Analysis of complex IL-35 confirmed its overexpression in RA (I) in comparison with OA (J) and PsA (K) synovial tissue (O). Corresponding synovial tissue sections stained with p35 (D), EBI3 (H) and IL-35 (L) IgG isotype controls. Original magnification 100. Bars represent mean ± SEM. ⁄ indicates comparisons among OA, RA and PsA, + indicates comparison between sublining and lining layer. P values less than 0.05 were considered statistically significant; ⁄/+ p < 0.05, ⁄⁄ /++ p< 0.01, ⁄⁄⁄/+++ p < 0.001.
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infiltrates in the RA synovial sublining layer, and the intensity varied from moderate to very strong. No or a weak expression pattern of both IL-35 subunits was detected in endothelial cells of the vessels in all synovial tissues. Semi-quantitative analysis (0–3 scale) showed that p35 staining intensity was significantly higher in RA compared with both OA and PsA synovial tissue sublining layer (1.43 ± 0.71 vs. 0.73 ± 0.58, vs. 0.53 ± 0.51; both p < 0.001, respectively). Furthermore, p35 was significantly upregulated in RA compared with PsA but not with OA synovial lining layer (1.50 ± 0.55 vs. 0.95 ± 0.22 vs. 1.33 ± 0.48; p < 0.001 and NS, respectively) (Fig. 1A–D, M). Similarly, the expression of EBI3 was significantly upregulated in RA compared with OA and PsA synovial sublining layer (1.75 ± 0.78 vs. 1.00 ± 0.53 vs. 0.45 ± 0.51; p < 0.01 and p < 0.001, respectively). In the lining layer, EBI3 was significantly upregulated in RA in comparison with OA but not PsA (1.35 ± 0.48 vs. 0.47 ± 0.51 vs. 1.10 ± 0.72; p < 0.001 and NS, respectively) (Fig. 1E–H, N). Analysis of the complex IL-35 molecule confirmed upregulation of IL-35 in RA compared to OA or PsA synovium (1.63 ± 0.74 vs. 0.20 ± 0.41 vs 0.05 ± 0.22; both p < 0.001 in the sublining layer and 0.47 ± 0.51 vs. 0.33 ± 0.48 vs. 0.15 ± 0.37; NS and p < 0.05, respectively in the lining layer) (Fig. 1I–L, O). The expression of IL-35 was more pronounced in RA sublining than in the lining layer (p < 0.001, Fig. 1I, O). Although the intensity of IL-35 staining was comparable with p35 and EBI3 subunits in the sublining layer, it appears less expressed in the lining layer which may be attributable to the epitope binding or
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sublining specific composition of inflammatory infiltrate as well as inflammatory environment. 3.2. IL-35 subunits (p35/EBI3) co-localize with immune cells and synovial fibroblasts To determine cell-specific localization of IL-35 subunits in synovial tissue samples, we performed double immunofluorescence staining using antibodies against p35, EBI3, and cell-specific markers for various cell types. Both IL-35 subunits were ubiquitously expressed in RA synovial tissue (Fig. 2). We demonstrated a colocalization of p35 (Fig. 2A) and EBI3 (Fig. 2B) subunits with markers specific for macrophages (CD68), dendritic cells (CD1a), nonepithelial cells of mesenchymal origin, mostly synovial fibroblasts (vimentin), and T cells (CD3) and B cells (CD20). 3.3. IL-35 expression in vitro is increased by inflammatory stimulation with TNFa To study the expression of subunits of IL-12 family cytokines in RASF (n = 6–12) and PBMCs (n = 8–12), the cells were stimulated with TNFa (10 ng/ml) for 6 and 24 h. We found that the expression of both p35 and EBI3 at mRNA level was significantly upregulated by TNFa (increased 3.5 fold and 165.7 fold after 24 h, respectively) in RASFs (Fig. 3A and B). Similarly to RASFs, treatment of PBMCs with TNFa (10 ng/ml) resulted in a time-dependent, although less-pronounced, induction of p35 and EBI3 mRNA expression
Fig. 2. Localization of both p35 (A) and EBI3 (B) subunits (in green) with cell-specific markers for various cell types such asB cells (CD20), T cells (CD3), macrophages (CD68), dendritic cells (CD1a), and non-epithelial cells of mesenchymal origin, mostly synovial fibroblasts (vimentin) (in red) in RA synovial tissue. Merge view is shown in the right panel.
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Fig. 3. Gene expression of interleukin (IL)-35 subunits (p35/EBI3) in RA synovial fibroblasts (RASFs) (A, B) and peripheral blood mononuclear cells (PBMCs) (C, D). IL-35 subunits p35 (A, C) and EBI3 (B, D) at the mRNA level were time-dependently induced by stimulation with tumor necrosis factor-(TNF)a. Bars represent mean ± SEM. P values less than 0.05 were considered statistically significant; ⁄ p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄ p < 0.001.
(1.6 fold and 4.9 fold after 24 h, respectively) (Fig. 3C and D). Expression of p40 and p28 was neither constitutively expressed nor induced by TNFa after 6 or 24 h (data not shown).
3.4. IL-35 induces expression of pro-inflammatory cytokines/ chemokines in PBMCs Gene expression analysis results obtained after 6 and 24 h showed upregulation of IL-1b (from 4.60 fold after IL-35 25 ng/ml to 6.24 fold after IL-35 100 ng/ml after 24 h) , IL-6 (from 2.58 to 9.57 fold), and MCP-1 (from 4.99 to 22.35 fold), but not TNFa (from 1.24 fold to 1.15 fold, data not shown). In agreement, we found that IL-35 dose-dependently induced release of IL-1b, IL-6, and MCP-1, but not TNFa, from PBMCs after 24 h of stimulation (Fig. 4). Moreover, treatment with a combination of both IL-35 and TNFa contributed to enhanced release of these cytokines from PBMCs into the cell culture supernatants (n = 6) after 24 h (Fig. 4). The O:E ratio indicated a synergistic effect of TNFa and IL-35 on the release of IL-1b (O:E ratio up to 2.3; p = 0.04) and IL-6 (O:E ratio up to 3.0; p = 0.006) and an additive effect on the release of MCP-1 (O:E ratio = 1.02; p = 0.99) from PBMCs. Stimulation of RASFs for 6 and 24 h, however, had no effect on the expression of the following genes: IL-1b, IL-6, TNFa, MCP-1, COX2, MMP-1, or MMP-3 (data not shown). Non-specific effect of IL-35 recombinant protein on PBMCs was excluded using proof-of- specificity experiments (Supplementary Fig. 1). To exclude any nonspecific effect of the recombinant protein mediated by the presence of the Fc IgG fragment or possible lipopolysaccharide (LPS) contamination, PBMCs (n = 3–6) were stimulated with IL-35 (100 ng/ml), polymyxin B sulfate (5 lg/ml), IL-35 (100 ng/ml) with polymyxin B sulfate (5 lg/ml), or control:Fc fusion human recombinant protein (100 ng/ml) for 24 h. No significant differences in levels of IL-1b, IL-6, MCP-1 or IL-8 were observed between controls and Fc fusion recombinant protein stimulated cells suggesting no effect of Fc IgG portion of recombinant IL-35 on release of abovementioned cytokines. Importantly, no LPS contamination was shown by comparable levels of IL-1b,
Fig. 4. Interleukin (IL)-35 dose-dependently induced release of IL-1b (A), IL-6 (B), and monocyte chemoattractant protein-1 (MCP-1) (C) from peripheral blood mononuclear cells. Combined stimulation with tumor necrosis factor-(TNF)a and IL-35 had a synergistic effect on the release of IL-1b (O:E ratio up to 2.3; p < 0.05) and IL-6 (O:E ratio up to 3.0; p < 0.01) and an additive effect on the release of MCP-1 (O:E ratio = 1.02; p = 1). Bars represent mean ± SEM. P values less than 0.05 were considered statistically significant; ⁄ p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄ p < 0.001.
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IL-6, MCP-1 or IL-8 in PBMCs stimulated with IL-35 alone or in combination with polymyxin B sulfate.
3.5. Expression of IL-35 receptor subunits in synovial tissue and its upregulation upon stimulation with TNFa We found the expression of both IL-35 receptor subunits IL12Rb2 and gp130 in RA, OA and PsA synovial tissues (Fig. 5). Although there was a trend towards higher expression of IL12Rb2 in RA synovium, the difference was not statistically significant (Fig. 5A–D, I). Intensity of gp130 staining in the synovium was more pronounced and revealed significant overexpression in RA compared with OA and PsA synovial tissue both in sublining as well as lining layers (2.10 ± 0.67 vs. 1.07 ± 0.52 vs. 0.70 ± 0.47; both p < 0.001 in the sublining layer and 1.83 ± 0.64 vs. 1.27 ± 0.45 vs.
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1.35 ± 0.49; p < 0.001 and p < 0.05, respectively in the lining layer) (Fig. 5E–H, J). In view of lack of the effect of IL-35 on RASFs, we analyzed the expression of IL-35 receptor subunits in RASFs and PBMCs in vitro. In RASFs, the expression of gp130 subunit was significantly upregulated by TNFa while the IL12Rb2 receptor subunit was expressed neither constitutively nor induced by TNFa (Fig. 6a). In contrast, PBMCs constitutively expressed both IL-35 receptor subunits. Unlike IL12Rb2, gp130 was not upregulated by TNFa after 24 h (Fig. 6b). Therefore, the unresponsiveness of RASF to IL-35 can be explained by the absence of IL-35 receptor on their surface. 4. Discussion In this study, we demonstrated for the first time an upregulation of the IL-35 in the synovial tissue in patients with RA com-
Fig. 5. Expression of IL-35 receptor subunits IL12Rb2 and gp130 in rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis (PsA) synovial tissues. Comparable expression of IL12Rb2 in RA (A), OA (B) and PsA (C) synovial tissues was revealed (I). Overexpression of gp130 in RA (E) compared with OA (F) and PsA (G) was identified (J). Corresponding synovial tissue sections stained with IL12Rb2 (D) and gp130 (H) IgG isotype controls. Original magnification 100. Bars represent mean±SEM. ⁄ indicates comparison among OA, RA and PsA, + indicates comparison between sublining and lining layer. P values less than 0.05 were considered statistically significant; ⁄/+ p < 0.05, ⁄⁄ /++ p < 0.01, ⁄⁄⁄/+++ p < 0.001.
Fig. 6. Analysis of IL-35 receptor subunits IL12Rb2 and gp130 revealed differential expression in RASFs (A) and PBMCs (B) and induction after stimulation with TNFa. Bars represent mean ± SEM. P values less than 0.05 were considered statistically significant; ⁄⁄ p < 0.01.
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pared with those who had OA or PsA. In addition, expression of both p35 and EBI3 subunits was induced upon TNFa stimulation in mononuclear cells and synovial fibroblasts. Moreover, IL-35 itself induced expression of several pro-inflammatory molecules in mononuclear cells that, unlike synovial fibroblasts, express both subunits of IL-35 receptor. While expression of IL12Rb2 receptor subunit is comparable among tissues, gp130 chain is significantly overexpressed in RA synovium. These data suggest pro-inflammatory properties of IL-35 in humans and a potential role for IL-35 in the pathogenesis of RA. This study is the first showing that both p35 and EBI3 subunits are upregulated in the lining as well as sublining layers of RA synovial tissue. IL-35 subunits were detected mostly in mononuclear inflammatory cells within the RA synovial sublining layer. Consistent with our findings, IL-12 (p35/p40)-positive cells have been localized in the synovial sublining layer in patients with RA [21]. The p19 subunit is abundantly expressed in the synovial lining layer at sites of invasion into cartilage and in vascular endothelium, while the p40 subunit is restricted to a few cells scattered in the synovium and is not co-expressed with p19 in synoviocytes [22]. There is evidence that the p35 subunit is constitutively expressed at low levels in a broad spectrum of cells [6]. EBI3 is expressed in placental trophoblasts, dendritic and plasma cells, macrophages, endothelial cells, Hodgkin and Reed-Sternberg lymphoma cells, and B cell lymphomas [23–28]. In agreement with these findings, we showed localization of the p35 and EBI3 subunits in macrophages, dendritic cells, B cells, T cells, and synovial fibroblasts in RA synovium. Our data, in keeping with others [22], show the absence of p40 and p28 in RASFs and support the hypothesis the only binding partner for p35 in RA synovial fibroblasts is the EBI3 subunit. Our study showed the induction of both p35 and EBI3 subunits in RA synovial fibroblasts and mononuclear cells after stimulation with TNFa. Consistently, both p35 and EBI3 subunits have been shown to be upregulated by pro-inflammatory stimuli such as TNFa, IFN-c, and TLR3 and TLR4 ligands in different cell types [24,29–31]. We hypothesize that the upregulation of p35/EBI3 in RA synovial tissue may be therefore attributable to induced expression of these subunits by chronic proinflammatory milieu as well as to synovial hyperplasia due to thickened lining layer and dense infiltration of inflammatory cells containing p35/EBI3 in RA synovium. While IL-12 and IL-23 are known as pro-inflammatory cytokines, the role of IL-27 is variable depending on the mouse model used [32–34]. The pro- or anti-inflammatory activities of IL-35 remain controversial and appear strongly dependent on experimental model and laboratory settings. IL-35 was shown to be expressed in mouse Treg cells in contrast to Teff cells and both subunits were required for maximal regulatory activity of Treg cells in a mouse model of inflammatory bowel disease [9]. Furthermore, administration of recombinant IL-35 attenuated collagen induced arthritis (CIA) in a mouse model [10,11]. The immunosuppressive potential of IL-35 in mice might be partially explained by its effect on the expansion of Treg cells and increased production of IL-10, suppressed Teff cell proliferation and inhibited differentiation of Th17 cells [9,10]. In addition, it was recently shown that IL-35 induces conversion of B cells to Breg cells that produce IL-35 as well as IL-10 [12]. IL-35 was shown to convert proliferative Foxp3-T cells into suppressive iTR35 cells that mediated their suppression exclusively via IL-35 and required no other suppressive cytokines (IL-10 or TGF-b) [11]. IL-35 also stimulated a new Treg CD39+ subset capable of controlling activated lymphocytes in a CIA model [13]. Although these data indicate anti-inflammatory properties of IL-35 in mouse models [9–11,13], other data have shown otherwise. Although the regulation of p35 in mouse and human Treg cells was similar, EBI3 gene expression appeared to substantially
differ. IL-35 expression has not been found in human Treg cells either constitutively or upon stimulation and is not required for their suppressive functions [7,16]. Interestingly, IL-35 failed to prevent the development of Lyme arthritis in mice but instead enhanced the inflammatory response in this model [14]. Moreover, introduction of IL-35 encoded plasmids exacerbated CIA in mice as it lead to Th17/Treg ratio imbalance and favored number of proinflammatory Th17 [15]. In keeping with these data, we demonstrated that like other IL-12 family members, IL-35 may rather have pro-inflammatory potential. We found that IL-35 induced release of several pro-inflammatory cytokines from mononuclear cells in vitro. The puzzle of IL-12 family members was completed by revealing IL-35 receptor that is composed IL12Rb2 and gp130, subunits shared with IL-12 and IL-27 receptor respectively [5]. Our data showed that while complex IL-35 receptor is present in mononuclear cells, it is absent in synovial fibroblasts. This fact may explain the lack of effect of IL-35 on RASFs. Our study is the first showing the overexpression of gp130 subunit of IL-35 receptor in RA synovium while the expression of IL12Rb2 was comparable across different tissues. We hypothesize that the regulation of IL12Rb2 and gp130 by TNFa, showed in our in vitro experiments and their differential expression in cell types may contribute to unique effect of IL-35 in RA synovium. 5. Conclusion In conclusion, we demonstrated increased expression of both IL-35 (p35/EBI3) subunits in RA synovial tissue that were localized in several immune cells and synovial fibroblasts. We showed regulation of IL-35 receptor chains by a pro-inflammatory mediator and differential expression of its gp130 subunit in RA synovium. Consistent with previous reports, we confirmed that IL-35 subunits are upregulated by TNFa. Furthermore, we found that IL-35 induces synthesis of some pro-inflammatory cytokines in mononuclear cells, which support that IL-35 is pro-inflammatory in RA. Funding This work was supported by Grant Project IGA No. 12440-4, SVV Project No. 264 511 and Project of Ministerstvo Zdravotnictví ˇ eské Republiky for conceptual development of research organizaC tion 023728. Acknowledgements The authors acknowledge Dr. Jakub Sikora for valuable help with confocal microscopy, Dr. Daniel Housa and Katerˇina Vadinská for assistance with immunohistochemistry, and Anna Kozáková and Marie Krabcová for technical assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2015.01.019. References [1] Klareskog L, Catrina AI, Paget S. Rheumatoid arthritis. Lancet 2009;373: 659–72. [2] Smolen JS, Aletaha D. The assessment of disease activity in rheumatoid arthritis. Clin Exp Rheumatol 2010;28:S18–27. [3] Senolt L, Vencovsky J, Pavelka K, Ospelt C, Gay S. Prospective new biological therapies for rheumatoid arthritis. Autoimmun Rev 2009;9:102–7. [4] Collison LW, Vignali DA. Interleukin-35: odd one out or part of the family? Immunol Rev 2008;226:248–62.
M. Filková et al. / Cytokine 73 (2015) 36–43 [5] Collison LW, Delgoffe GM, Guy CS, Vignali KM, Chaturvedi V, Fairweather D, et al. The composition and signaling of the IL-35 receptor are unconventional. Nat Immunol 2012;13:290–9. [6] Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3:133–46. [7] Bardel E, Larousserie F, Charlot-Rabiega P, Coulomb-L’Hermine A, Devergne O. Human CD4+ CD25+ Foxp3+ regulatory T cells do not constitutively express IL35. J Immunol 2008;181:6898–905. [8] Devergne O, Birkenbach M, Kieff E. Epstein-Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin. Proc Natl Acad Sci USA 1997;94:12041–6. [9] Collison LW, Workman CJ, Kuo TT, Boyd K, Wang Y, Vignali KM, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007;450:566–9. [10] Niedbala W, Wei XQ, Cai B, Hueber AJ, Leung BP, McInnes IB, et al. IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells. Eur J Immunol 2007;37:3021–9. [11] Collison LW, Chaturvedi V, Henderson AL, Giacomin PR, Guy C, Bankoti J, et al. IL-35-mediated induction of a potent regulatory T cell population. Nat Immunol 2010;11:1093–101. [12] Wang RX, Yu CR, Dambuza IM, Mahdi RM, Dolinska MB, Sergeev YV, et al. Interleukin-35 induces regulatory B cells that suppress autoimmune disease. Nat Med 2014;20:633–41. [13] Kochetkova I, Golden S, Holderness K, Callis G, Pascual DW. IL-35 stimulation of CD39+ regulatory T cells confers protection against collagen II-induced arthritis via the production of IL-10. J Immunol 2010;184:7144–53. [14] Kuo J, Nardelli DT, Warner TF, Callister SM, Schell RF. Interleukin-35 enhances Lyme arthritis in Borrelia-vaccinated and -infected mice. Clin Vaccine Immunol: CVI 2011;18:1125–32. [15] Thiolat A, Denys A, Petit M, Biton J, Lemeiter D, Herve R, et al. Interleukin-35 gene therapy exacerbates experimental rheumatoid arthritis in mice. Cytokine 2014;69:87–93. [16] Allan SE, Song-Zhao GX, Abraham T, McMurchy AN, Levings MK. Inducible reprogramming of human T cells into Treg cells by a conditionally active form of FOXP3. Eur J Immunol 2008;38:3282–9. [17] Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. [18] Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, et al. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and therapeutic criteria committee of the American Rheumatism Association. Arthritis Rheum 1986;29:1039–49. [19] Taylor W, Gladman D, Helliwell P, Marchesoni A, Mease P, Mielants H, et al. Classification criteria for psoriatic arthritis: development of new criteria from a large international study. Arthritis Rheum 2006;54:2665–73.
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[20] Senolt L, Housa D, Vernerova Z, Jirasek T, Svobodova R, Veigl D, et al. Resistin in rheumatoid arthritis synovial tissue, synovial fluid and serum. Ann Rheum Dis 2007;66:458–63. [21] Morita Y, Yamamura M, Nishida K, Harada S, Okamoto H, Inoue H, et al. Expression of interleukin-12 in synovial tissue from patients with rheumatoid arthritis. Arthritis Rheum 1998;41:306–14. [22] Brentano F, Ospelt C, Stanczyk J, Gay RE, Gay S, Kyburz D. Abundant expression of the interleukin (IL)23 subunit p19, but low levels of bioactive IL23 in the rheumatoid synovium: differential expression and Toll-like receptor-(TLR) dependent regulation of the IL23 subunits, p19 and p40, in rheumatoid arthritis. Ann Rheum Dis 2009;68:143–50. [23] Devergne O, Coulomb-L’Hermine A, Capel F, Moussa M, Capron F. Expression of Epstein–Barr virus-induced gene 3, an interleukin-12 p40-related molecule, throughout human pregnancy: involvement of syncytiotrophoblasts and extravillous trophoblasts. Am J Pathol 2001;159:1763–76. [24] Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, et al. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4+ T cells. Immunity 2002;16:779–90. [25] Kempe S, Kestler H, Lasar A, Wirth T. NF-kappaB controls the global proinflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program. Nucleic Acids Res 2005;33:5308–19. [26] Larousserie F, Pflanz S, Coulomb-L’Hermine A, Brousse N, Kastelein R, Devergne O. Expression of IL-27 in human Th1-associated granulomatous diseases. J Pathol 2004;202:164–71. [27] Niedobitek G, Pazolt D, Teichmann M, Devergne O. Frequent expression of the Epstein–Barr virus (EBV)-induced gene, EBI3, an IL-12 p40-related cytokine, in Hodgkin and Reed-Sternberg cells. J Pathol 2002;198:310–6. [28] Larousserie F, Bardel E, Coulomb L’Hermine A, Canioni D, Brousse N, Kastelein RA, et al. Variable expression of Epstein–Barr virus-induced gene 3 during normal B-cell differentiation and among B-cell lymphomas. J Pathol 2006;209:360–8. [29] Kempe S, Heinz P, Kokai E, Devergne O, Marx N, Wirth T. Epstein-barr virusinduced gene-3 is expressed in human atheroma plaques. Am J Pathol 2009;175:440–7. [30] Goriely S, Molle C, Nguyen M, Albarani V, Haddou NO, Lin R, et al. Interferon regulatory factor 3 is involved in Toll-like receptor 4 (TLR4)- and TLR3-induced IL-12p35 gene activation. Blood 2006;107:1078–84. [31] Wirtz S, Becker C, Fantini MC, Nieuwenhuis EE, Tubbe I, Galle PR, et al. EBVinduced gene 3 transcription is induced by TLR signaling in primary dendritic cells via NF-kappa B activation. J Immunol 2005;174:2814–24. [32] Niedbala W, Cai B, Wei X, Patakas A, Leung BP, McInnes IB, et al. Interleukin 27 attenuates collagen-induced arthritis. Ann Rheum Dis 2008;67:1474–9. [33] Cao Y, Doodes PD, Glant TT, Finnegan A. IL-27 induces a Th1 immune response and susceptibility to experimental arthritis. J Immunol 2008;180:922–30. [34] Rajaiah R, Puttabyatappa M, Polumuri SK, Moudgil KD. Interleukin-27 and interferon-gamma are involved in regulation of autoimmune arthritis. J Biol Chem 2011;286:2817–25.
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Pro-inflammatory effects of interleukin-35 in rheumatoid arthritis Mária Filková a,⇑, Zdenka Vernerová b, Hana Hulejová a, Klára Prajzlerová a, David Veigl c, Karel Pavelka a,d, Jirˇí Vencovsky´ a,d, Ladislav Šenolt a,d a
Institute of Rheumatology, Prague, Czech Republic Institute of Pathology of the 3rd Faculty of Medicine, Charles University in Prague, Prague, Czech Republic c 1st Orthopedic Clinic, 1st Faculty of Medicine and Faculty Hospital Motol, Charles University in Prague, Prague, Czech Republic d Department of Rheumatology, 1st Faculty of Medicine, Charles University in Prague, Czech Republic b
a r t i c l e
i n f o
Article history: Received 16 March 2014 Received in revised form 30 October 2014 Accepted 23 January 2015
Keywords: Rheumatoid arthritis Synovial tissue Inflammation Interleukin-35
a b s t r a c t Objective: Interleukin-35 (IL-35) is a heterodimeric member of the IL-12 family consisting of p35/IL-12a and EBI3/IL-27b subunits. Expressed in murine Treg cells, IL-35 controls inflammatory diseases in mouse models. However, human IL-35 is expressed in Teff cells rather than in Treg cells and is shown to be upregulated under inflammatory conditions. Our aim was to examine the involvement of IL-35 in the pathogenesis of rheumatoid arthritis (RA). Methods: Immunohistochemical and immunofluorescence analysis was used to determine the expression and localization of IL-35 and its subunits (p35/EBI3) and IL-35 receptor (IL12Rb2/gp130) in RA, osteoarthritis (OA) and psoriatic arthritis (PsA) synovial tissues. Expression of p35/EBI3 subunits and release of inflammatory cytokines upon stimulation with IL-35 were assessed in RA synovial fibroblasts (SFs) and peripheral blood mononuclear cells (PBMCs). Results: Both IL-35 and its subunits were upregulated in RA in comparison with OA or PsA synovium. Using cell-specific markers, p35 and EBI3 were identified in macrophages, dendritic cells, SFs, and T as well as B cells in RA synovium. Both p35 and EBI3 were induced by TNFa in RASFs and PBMCs. IL-35 dose-dependently upregulated release of pro-inflammatory mediators IL-1b, IL-6 and MCP-1 in PBMCs. While gp130 receptor subunit was upregulated in RA synovium and was expressed in RASFs and PBMCs, there was no difference in IL12Rb2 expression subunit among tissues and its presence in RASFs was lacking. Conclusion: Upregulation of IL-35 at sites of inflammation in RA and its pro-inflammatory potential suggests that IL-35 might play an important role in RA pathogenesis. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation and hyperplasia of the synovial tissue, increased production of inflammatory mediators, and subsequent joint destruction. It is now well established that macrophages, activated T and B cells, and their products including tumor necrosis factor-a (TNFa) and interleukin (IL)-1b and IL-6 play a significant role in the pathogenesis of RA [1]. Although currently available biologic treatment targets these specific cellular and molecular mechanisms, only a minority of patients achieve remission [2]. Therefore, studies of various molecular pathways have focused on targets for future therapies [3].
⇑ Corresponding author at: Institute of Rheumatology, Na Slupi 4, Prague 128 50, Czech Republic. Tel.: +420 234 075 323; fax: +420 224 914 451. E-mail address: [email protected] (M. Filková). http://dx.doi.org/10.1016/j.cyto.2015.01.019 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
IL-35 is a newly described cytokine that belongs to the IL-12 family consisting of the heterodimeric cytokines IL-12, IL-23, IL-27, and IL-35 [4]. Similar to other IL-12 members, IL-35 consists of a chain (p35/IL-12a) that can also dimerize with p40 to form IL-12, and b chain (EBI3/IL-27b) sharing a partnership with the p28 subunit of IL-27. Like the cytokine chains, also subunits of IL-35 receptor are shared among IL-12 family members. IL-35 receptor consists of IL12Rb2 (IL-12 receptor component) and gp130 (IL-27 receptor component) activating subsequently STAT1 and STAT4 signaling pathways [5]. While p35 is ubiquitously expressed, EBI3 (Epstein–Barr virusinduced gene 3) is selectively produced and is highly inducible [6,7]. Although the association between p35 and EBI3 was described several years ago, the biologic role of this heterodimer has remained undetermined until now [8]. However, data on animal models remain controversial. While some support anti-inflammatory activities of IL-35 in inflammatory bowel disease, collagen induced arthritis (CIA) or other autoimmune conditions given its
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effect on Treg, Breg and associated cytokines [9–13], other data are suggestive of its pro-inflammatory properties. IL-35 was shown to act as an inflammatory mediator in Lyme arthritis or CIA under different experimental settings [14,15]. Recently, IL-35 was shown to convert proliferative Foxp3-T cells into suppressive iTR35 cells that mediated their suppression exclusively via IL-35 and to stimulate a new Treg CD39+ subset capable of controlling activated lymphocytes in a CIA model [11,13]. There is evidence that EBI3 is a downstream target of Foxp3 in mouse Treg cells [9], however, induction of Foxp3 activity does not upregulate EBI3 or p35 in humans [16]. These data show that IL-35 is not exclusively an anti-inflammatory cytokine and its role in different inflammatory animal models or human Treg cells remains contradictive [7]. Therefore IL-35 may play different roles in mice and humans. These reports bring firm evidence that IL-35 plays a role in autoimmune inflammatory diseases. Therefore, the aim of this study was to analyze the expression of IL-35 and its subunits in synovial tissue of patients with RA, psoriatic arthritis (PsA) and osteoarthritis (OA), and to investigate possible involvement of IL35 in the pathogenesis of RA.
2. Methods 2.1. Immunohistochemistry Synovial tissue specimens were obtained during reconstructive hand surgery or wrist synovectomy in patients with RA (n = 5), during total knee joint prosthesis in patients with OA (n = 5) and PsA (n = 2). Patients with RA fulfilled the 1987 revised criteria of the American College of Rheumatology (ACR), patients with OA met the ACR criteria for OA of the knee and patients with PsA met CASPAR diagnostic criteria [17–19]. Written informed consent from each participant was obtained prior to enrollment, and the study was approved by the local ethics committee at the Institute of Rheumatology in Prague. Formaldehyde-fixed, paraffin-embedded sections (5 lm) were deparaffinized and rehydrated. Endogenous peroxidase activity was inhibited by 3% hydrogen peroxide in methanol for 30 min followed by 15 min of rinsing in tap water. Non-specific activity was avoided by pretreatment of sections with 1% normal bovine serum in phosphate-buffered saline (PBS) for 2 h. Immunohistochemical labeling was performed after antigen retrieval in Target retrieval solution pH 6.0 (Dako, Cytomation, Glostrup, Denmark) for 30 min at 98 °C and slides were incubated for 1 h at room temperature either with rabbit polyclonal anti-human CD130 antibody (Novus Biologicals, Littleton, CO, USA) diluted 1:2000, with rabbit polyclonal anti-human IL-12a antibody (Abcam, Cambridge, UK) diluted 1:4000, or with mouse monoclonal anti-human antibody to IL-35 (clone 15K8D10, Acris antibodies, San Diego, CA, USA) diluted 1:2000. All antibodies were diluted in the ChemMate antibody diluent (Dako, Cytomation, Glostrup, Denmark). Another immunohistochemical labeling was performed after antigen retrieval in Target retrieval solution pH 9.0 (Dako, Cytomation, Glostrup, Denmark), for 30 min at 98 °C and slides were incubated 1 hour at room temperature either with rabbit polyclonal anti-human IL12RB2 antibody (Bioss antibodies, Woburn, Massachusetts, USA) diluted 1:300 in ChemMate antibody diluent (Dako, Cytomation, Glostrup, Denmark), or rabbit polyclonal anti-human EBI3 antibody (Lifespan Biosciences, Seattle, WA, USA) diluted 1:100 in the same antibody diluent. The Envision kit (Dako, Cytomation) was used to visualize positive slides. The chromogen 3,3-diaminobenzidine (Liquid DAB + Substrate, Dako, Cytomation) was applied to all sections, and counterstaining was performed with Mayer’s hematoxylin. Isotype IgG (Dako, Cytomation) was used
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as a negative control. All slides were analyzed semi-quantitatively using a Nikon Eclipse E600 microscope (Nikon, Melville, NY, USA) in a blind fashion by two independent readers. The analysis included ten different fields throughout the biopsies separately in both lining and sublining layers (magnification 400), and a global score was assigned using a four-point scale: 0 represented negative staining intensity, and scores of 1–3 represented weak, moderate, and strong staining intensity, respectively. 2.2. Laser scanning confocal microscopy Confocal microscopy double immunolabeling was performed as follows: Slides were simultaneously incubated after antigen retrieval in 0.1 mol/l citrate buffer, pH 6.0, for 40 min with primary mouse anti-human CD20 monoclonal antibodies, clone L26, anti-human CD68 monoclonal antibodies, clone PG-M1, anti-human CD1a monoclonal antibodies, clone O1; anti-human CD3 monoclonal antibody, clone PC3; rabbit anti-human CD3 polyclonal antibodies or mouse anti-human vimentin monoclonal antibodies, clone 3B4 (all Dako, Cytomation), respectively. Further, we used primary rabbit anti-human EBI3 polyclonal antibodies (Lifespan Biosciences) or mouse anti-human IL-12a monoclonal antibodies (Santa Cruz, Santa Cruz, CA, USA). After 1 h, simultaneous incubation of the specimen was performed with FITC/CY5–conjugated secondary antibodiesAlexa Fluor 488 goat anti-mouse IgG (Invitrogen, Basel, Switzerland) and Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen) in the case of EBI3. In the case of IL-12a, we used FITC/CY5– conjugated secondary antibodies Alexa Fluor 488 goat anti-mouse IgG2 or IgG3 (Invitrogen) or Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 568 goat anti-mouse IgG1 (Invitrogen). All of these antibodies were diluted in PBS and all incubations took place at 20 °C for 30 min. The slides were mounted in Mowiol (Hoechst, Frankfurt am Main, Germany) and analyzed using a confocal laser scanning microscope, the Nikon Eclipse TE2000 (Nikon). Simultaneous excitation with anargon–krypton laser at wavelengths of 488 nm and 548 nm and appropriate 515 ± 15 and 590 ± 15 nm band-pass fluorescence filters were used for excitation and detection (in separate channel detection mode), respectively. 2.3. Cell cultures and stimulation assays Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll density gradient centrifugation from blood samples from healthy donors. Freshly isolated PBMCs were incubated in advanced RPMI medium 1640 (Invitrogen) supplemented with Lglutamine (Gibco, Carlsbad, CA, USA) and then stimulated for 6 or 24 h with the following agents: TNFa (10 ng/ml; R&D Systems, Abingdon, United Kingdom), IL-35:Fc human recombinant protein (25, 50, 100 ng/ml; ALEXIS Biochemicals, Enzo Life Sciences, Lausen, Switzerland), control: Fc fusion human recombinant protein (100 ng/ml; ALEXIS Biochemicals, Enzo Life Sciences), and polymyxin B sulfate (5 lg/ml; Sigma–Aldrich, St. Louis, MO, USA) at 37 °C in a 5% CO2 humidified atmosphere. RA synovial fibroblasts were isolated from synovial tissues as described previously [20] and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal calf serum (FCS) at 37 °C in a 5% CO2 humidified atmosphere. Synovial fibroblasts between passages 4 and 8 were stimulated with TNFa (10 ng/ml) or IL-35 (25, 50, 100 ng/ml) for 6 and 24 h. Cells were lysed in RLT buffer (QIAGEN, Hilden, Germany) after 6 and 24 h, and cell culture supernatants were collected after 24 h. Samples were stored at 80 °C until use. 2.4. RT-PCR analysis Total RNA was isolated using a MagNA Pure Compact RNA Isolation Kit for the MagNA Pure Compact Instrument (Roche, Mann-
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heim, Germany), and reverse transcription was performed with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). For real-time polymerase chain reaction (RT-PCR), TaqMan gene expression assays (Applied Biosystems) were used, and the reaction was performed with a 7900HT Fast Real-Time PCR System (Applied Biosystems). Data were analyzed using the dCt method for relative quantification with 18S as an endogenous control. 2.5. ELISA Cytokines IL-1b, IL-6, TNFa and monocyte chemoattractant protein-1 (MCP-1) released into the cell culture supernatants were measured by ELISA kit (RayBiotech, Norcross, GA,USA) and IL-8 was measured using IL-8 DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA). The absorbance was measured at 450 nm using an ELISA reader (Tecan Sunrise, Salzburg, Austria). 2.6. Statistical analysis The Mann–Whitney U-test, paired t-test, Kruskal–Wallis and Friedman test including Dunn’s multiple comparisons were used
where appropriate. To calculate the additive or synergistic effect of combined treatment of IL-35 plus TNFa on the expression of cytokines/chemokines, the observed (O; concentrations obtained for the combination of TNFa and IL-35) to expected (E; sum of the independent concentrations obtained for TNFa and IL-35, respectively) ratio (O:E) was calculated. The significance of differences between observed and expected concentrations was determined by paired t-tests. P values less than 0.05 were considered statistically significant. The analysis was performed and graphs created using GraphPad Prism 5 (version 5.02; GraphPad Software, La Jolla, CA, USA). 3. Results 3.1. IL-35 and its subunits (p35/EBI3) are upregulated in RA synovial tissue We found that the expression of both IL-35 subunits p35 and EBI3 in synovial tissue was present in patients with RA and that the staining intensity was significantly enhanced compared with patients with OA or PsA (Fig. 1). There was consistent expression of both IL-35 subunits within the mononuclear inflammatory
Fig. 1. Expression of p35 and EBI3 subunits of IL-35 in rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis (PsA) synovial tissues. Higher expression of p35 in RA (A) than in OA (B) and PsA (C) synovial tissue was demonstrated by semi-quantitative analysis (M). Similarly, overexpression of EBI3 in RA (E) compared with OA (F) and PsA (G) synovial tissue was revealed (N). Analysis of complex IL-35 confirmed its overexpression in RA (I) in comparison with OA (J) and PsA (K) synovial tissue (O). Corresponding synovial tissue sections stained with p35 (D), EBI3 (H) and IL-35 (L) IgG isotype controls. Original magnification 100. Bars represent mean ± SEM. ⁄ indicates comparisons among OA, RA and PsA, + indicates comparison between sublining and lining layer. P values less than 0.05 were considered statistically significant; ⁄/+ p < 0.05, ⁄⁄ /++ p< 0.01, ⁄⁄⁄/+++ p < 0.001.
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infiltrates in the RA synovial sublining layer, and the intensity varied from moderate to very strong. No or a weak expression pattern of both IL-35 subunits was detected in endothelial cells of the vessels in all synovial tissues. Semi-quantitative analysis (0–3 scale) showed that p35 staining intensity was significantly higher in RA compared with both OA and PsA synovial tissue sublining layer (1.43 ± 0.71 vs. 0.73 ± 0.58, vs. 0.53 ± 0.51; both p < 0.001, respectively). Furthermore, p35 was significantly upregulated in RA compared with PsA but not with OA synovial lining layer (1.50 ± 0.55 vs. 0.95 ± 0.22 vs. 1.33 ± 0.48; p < 0.001 and NS, respectively) (Fig. 1A–D, M). Similarly, the expression of EBI3 was significantly upregulated in RA compared with OA and PsA synovial sublining layer (1.75 ± 0.78 vs. 1.00 ± 0.53 vs. 0.45 ± 0.51; p < 0.01 and p < 0.001, respectively). In the lining layer, EBI3 was significantly upregulated in RA in comparison with OA but not PsA (1.35 ± 0.48 vs. 0.47 ± 0.51 vs. 1.10 ± 0.72; p < 0.001 and NS, respectively) (Fig. 1E–H, N). Analysis of the complex IL-35 molecule confirmed upregulation of IL-35 in RA compared to OA or PsA synovium (1.63 ± 0.74 vs. 0.20 ± 0.41 vs 0.05 ± 0.22; both p < 0.001 in the sublining layer and 0.47 ± 0.51 vs. 0.33 ± 0.48 vs. 0.15 ± 0.37; NS and p < 0.05, respectively in the lining layer) (Fig. 1I–L, O). The expression of IL-35 was more pronounced in RA sublining than in the lining layer (p < 0.001, Fig. 1I, O). Although the intensity of IL-35 staining was comparable with p35 and EBI3 subunits in the sublining layer, it appears less expressed in the lining layer which may be attributable to the epitope binding or
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sublining specific composition of inflammatory infiltrate as well as inflammatory environment. 3.2. IL-35 subunits (p35/EBI3) co-localize with immune cells and synovial fibroblasts To determine cell-specific localization of IL-35 subunits in synovial tissue samples, we performed double immunofluorescence staining using antibodies against p35, EBI3, and cell-specific markers for various cell types. Both IL-35 subunits were ubiquitously expressed in RA synovial tissue (Fig. 2). We demonstrated a colocalization of p35 (Fig. 2A) and EBI3 (Fig. 2B) subunits with markers specific for macrophages (CD68), dendritic cells (CD1a), nonepithelial cells of mesenchymal origin, mostly synovial fibroblasts (vimentin), and T cells (CD3) and B cells (CD20). 3.3. IL-35 expression in vitro is increased by inflammatory stimulation with TNFa To study the expression of subunits of IL-12 family cytokines in RASF (n = 6–12) and PBMCs (n = 8–12), the cells were stimulated with TNFa (10 ng/ml) for 6 and 24 h. We found that the expression of both p35 and EBI3 at mRNA level was significantly upregulated by TNFa (increased 3.5 fold and 165.7 fold after 24 h, respectively) in RASFs (Fig. 3A and B). Similarly to RASFs, treatment of PBMCs with TNFa (10 ng/ml) resulted in a time-dependent, although less-pronounced, induction of p35 and EBI3 mRNA expression
Fig. 2. Localization of both p35 (A) and EBI3 (B) subunits (in green) with cell-specific markers for various cell types such asB cells (CD20), T cells (CD3), macrophages (CD68), dendritic cells (CD1a), and non-epithelial cells of mesenchymal origin, mostly synovial fibroblasts (vimentin) (in red) in RA synovial tissue. Merge view is shown in the right panel.
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Fig. 3. Gene expression of interleukin (IL)-35 subunits (p35/EBI3) in RA synovial fibroblasts (RASFs) (A, B) and peripheral blood mononuclear cells (PBMCs) (C, D). IL-35 subunits p35 (A, C) and EBI3 (B, D) at the mRNA level were time-dependently induced by stimulation with tumor necrosis factor-(TNF)a. Bars represent mean ± SEM. P values less than 0.05 were considered statistically significant; ⁄ p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄ p < 0.001.
(1.6 fold and 4.9 fold after 24 h, respectively) (Fig. 3C and D). Expression of p40 and p28 was neither constitutively expressed nor induced by TNFa after 6 or 24 h (data not shown).
3.4. IL-35 induces expression of pro-inflammatory cytokines/ chemokines in PBMCs Gene expression analysis results obtained after 6 and 24 h showed upregulation of IL-1b (from 4.60 fold after IL-35 25 ng/ml to 6.24 fold after IL-35 100 ng/ml after 24 h) , IL-6 (from 2.58 to 9.57 fold), and MCP-1 (from 4.99 to 22.35 fold), but not TNFa (from 1.24 fold to 1.15 fold, data not shown). In agreement, we found that IL-35 dose-dependently induced release of IL-1b, IL-6, and MCP-1, but not TNFa, from PBMCs after 24 h of stimulation (Fig. 4). Moreover, treatment with a combination of both IL-35 and TNFa contributed to enhanced release of these cytokines from PBMCs into the cell culture supernatants (n = 6) after 24 h (Fig. 4). The O:E ratio indicated a synergistic effect of TNFa and IL-35 on the release of IL-1b (O:E ratio up to 2.3; p = 0.04) and IL-6 (O:E ratio up to 3.0; p = 0.006) and an additive effect on the release of MCP-1 (O:E ratio = 1.02; p = 0.99) from PBMCs. Stimulation of RASFs for 6 and 24 h, however, had no effect on the expression of the following genes: IL-1b, IL-6, TNFa, MCP-1, COX2, MMP-1, or MMP-3 (data not shown). Non-specific effect of IL-35 recombinant protein on PBMCs was excluded using proof-of- specificity experiments (Supplementary Fig. 1). To exclude any nonspecific effect of the recombinant protein mediated by the presence of the Fc IgG fragment or possible lipopolysaccharide (LPS) contamination, PBMCs (n = 3–6) were stimulated with IL-35 (100 ng/ml), polymyxin B sulfate (5 lg/ml), IL-35 (100 ng/ml) with polymyxin B sulfate (5 lg/ml), or control:Fc fusion human recombinant protein (100 ng/ml) for 24 h. No significant differences in levels of IL-1b, IL-6, MCP-1 or IL-8 were observed between controls and Fc fusion recombinant protein stimulated cells suggesting no effect of Fc IgG portion of recombinant IL-35 on release of abovementioned cytokines. Importantly, no LPS contamination was shown by comparable levels of IL-1b,
Fig. 4. Interleukin (IL)-35 dose-dependently induced release of IL-1b (A), IL-6 (B), and monocyte chemoattractant protein-1 (MCP-1) (C) from peripheral blood mononuclear cells. Combined stimulation with tumor necrosis factor-(TNF)a and IL-35 had a synergistic effect on the release of IL-1b (O:E ratio up to 2.3; p < 0.05) and IL-6 (O:E ratio up to 3.0; p < 0.01) and an additive effect on the release of MCP-1 (O:E ratio = 1.02; p = 1). Bars represent mean ± SEM. P values less than 0.05 were considered statistically significant; ⁄ p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄ p < 0.001.
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IL-6, MCP-1 or IL-8 in PBMCs stimulated with IL-35 alone or in combination with polymyxin B sulfate.
3.5. Expression of IL-35 receptor subunits in synovial tissue and its upregulation upon stimulation with TNFa We found the expression of both IL-35 receptor subunits IL12Rb2 and gp130 in RA, OA and PsA synovial tissues (Fig. 5). Although there was a trend towards higher expression of IL12Rb2 in RA synovium, the difference was not statistically significant (Fig. 5A–D, I). Intensity of gp130 staining in the synovium was more pronounced and revealed significant overexpression in RA compared with OA and PsA synovial tissue both in sublining as well as lining layers (2.10 ± 0.67 vs. 1.07 ± 0.52 vs. 0.70 ± 0.47; both p < 0.001 in the sublining layer and 1.83 ± 0.64 vs. 1.27 ± 0.45 vs.
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1.35 ± 0.49; p < 0.001 and p < 0.05, respectively in the lining layer) (Fig. 5E–H, J). In view of lack of the effect of IL-35 on RASFs, we analyzed the expression of IL-35 receptor subunits in RASFs and PBMCs in vitro. In RASFs, the expression of gp130 subunit was significantly upregulated by TNFa while the IL12Rb2 receptor subunit was expressed neither constitutively nor induced by TNFa (Fig. 6a). In contrast, PBMCs constitutively expressed both IL-35 receptor subunits. Unlike IL12Rb2, gp130 was not upregulated by TNFa after 24 h (Fig. 6b). Therefore, the unresponsiveness of RASF to IL-35 can be explained by the absence of IL-35 receptor on their surface. 4. Discussion In this study, we demonstrated for the first time an upregulation of the IL-35 in the synovial tissue in patients with RA com-
Fig. 5. Expression of IL-35 receptor subunits IL12Rb2 and gp130 in rheumatoid arthritis (RA), osteoarthritis (OA) and psoriatic arthritis (PsA) synovial tissues. Comparable expression of IL12Rb2 in RA (A), OA (B) and PsA (C) synovial tissues was revealed (I). Overexpression of gp130 in RA (E) compared with OA (F) and PsA (G) was identified (J). Corresponding synovial tissue sections stained with IL12Rb2 (D) and gp130 (H) IgG isotype controls. Original magnification 100. Bars represent mean±SEM. ⁄ indicates comparison among OA, RA and PsA, + indicates comparison between sublining and lining layer. P values less than 0.05 were considered statistically significant; ⁄/+ p < 0.05, ⁄⁄ /++ p < 0.01, ⁄⁄⁄/+++ p < 0.001.
Fig. 6. Analysis of IL-35 receptor subunits IL12Rb2 and gp130 revealed differential expression in RASFs (A) and PBMCs (B) and induction after stimulation with TNFa. Bars represent mean ± SEM. P values less than 0.05 were considered statistically significant; ⁄⁄ p < 0.01.
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pared with those who had OA or PsA. In addition, expression of both p35 and EBI3 subunits was induced upon TNFa stimulation in mononuclear cells and synovial fibroblasts. Moreover, IL-35 itself induced expression of several pro-inflammatory molecules in mononuclear cells that, unlike synovial fibroblasts, express both subunits of IL-35 receptor. While expression of IL12Rb2 receptor subunit is comparable among tissues, gp130 chain is significantly overexpressed in RA synovium. These data suggest pro-inflammatory properties of IL-35 in humans and a potential role for IL-35 in the pathogenesis of RA. This study is the first showing that both p35 and EBI3 subunits are upregulated in the lining as well as sublining layers of RA synovial tissue. IL-35 subunits were detected mostly in mononuclear inflammatory cells within the RA synovial sublining layer. Consistent with our findings, IL-12 (p35/p40)-positive cells have been localized in the synovial sublining layer in patients with RA [21]. The p19 subunit is abundantly expressed in the synovial lining layer at sites of invasion into cartilage and in vascular endothelium, while the p40 subunit is restricted to a few cells scattered in the synovium and is not co-expressed with p19 in synoviocytes [22]. There is evidence that the p35 subunit is constitutively expressed at low levels in a broad spectrum of cells [6]. EBI3 is expressed in placental trophoblasts, dendritic and plasma cells, macrophages, endothelial cells, Hodgkin and Reed-Sternberg lymphoma cells, and B cell lymphomas [23–28]. In agreement with these findings, we showed localization of the p35 and EBI3 subunits in macrophages, dendritic cells, B cells, T cells, and synovial fibroblasts in RA synovium. Our data, in keeping with others [22], show the absence of p40 and p28 in RASFs and support the hypothesis the only binding partner for p35 in RA synovial fibroblasts is the EBI3 subunit. Our study showed the induction of both p35 and EBI3 subunits in RA synovial fibroblasts and mononuclear cells after stimulation with TNFa. Consistently, both p35 and EBI3 subunits have been shown to be upregulated by pro-inflammatory stimuli such as TNFa, IFN-c, and TLR3 and TLR4 ligands in different cell types [24,29–31]. We hypothesize that the upregulation of p35/EBI3 in RA synovial tissue may be therefore attributable to induced expression of these subunits by chronic proinflammatory milieu as well as to synovial hyperplasia due to thickened lining layer and dense infiltration of inflammatory cells containing p35/EBI3 in RA synovium. While IL-12 and IL-23 are known as pro-inflammatory cytokines, the role of IL-27 is variable depending on the mouse model used [32–34]. The pro- or anti-inflammatory activities of IL-35 remain controversial and appear strongly dependent on experimental model and laboratory settings. IL-35 was shown to be expressed in mouse Treg cells in contrast to Teff cells and both subunits were required for maximal regulatory activity of Treg cells in a mouse model of inflammatory bowel disease [9]. Furthermore, administration of recombinant IL-35 attenuated collagen induced arthritis (CIA) in a mouse model [10,11]. The immunosuppressive potential of IL-35 in mice might be partially explained by its effect on the expansion of Treg cells and increased production of IL-10, suppressed Teff cell proliferation and inhibited differentiation of Th17 cells [9,10]. In addition, it was recently shown that IL-35 induces conversion of B cells to Breg cells that produce IL-35 as well as IL-10 [12]. IL-35 was shown to convert proliferative Foxp3-T cells into suppressive iTR35 cells that mediated their suppression exclusively via IL-35 and required no other suppressive cytokines (IL-10 or TGF-b) [11]. IL-35 also stimulated a new Treg CD39+ subset capable of controlling activated lymphocytes in a CIA model [13]. Although these data indicate anti-inflammatory properties of IL-35 in mouse models [9–11,13], other data have shown otherwise. Although the regulation of p35 in mouse and human Treg cells was similar, EBI3 gene expression appeared to substantially
differ. IL-35 expression has not been found in human Treg cells either constitutively or upon stimulation and is not required for their suppressive functions [7,16]. Interestingly, IL-35 failed to prevent the development of Lyme arthritis in mice but instead enhanced the inflammatory response in this model [14]. Moreover, introduction of IL-35 encoded plasmids exacerbated CIA in mice as it lead to Th17/Treg ratio imbalance and favored number of proinflammatory Th17 [15]. In keeping with these data, we demonstrated that like other IL-12 family members, IL-35 may rather have pro-inflammatory potential. We found that IL-35 induced release of several pro-inflammatory cytokines from mononuclear cells in vitro. The puzzle of IL-12 family members was completed by revealing IL-35 receptor that is composed IL12Rb2 and gp130, subunits shared with IL-12 and IL-27 receptor respectively [5]. Our data showed that while complex IL-35 receptor is present in mononuclear cells, it is absent in synovial fibroblasts. This fact may explain the lack of effect of IL-35 on RASFs. Our study is the first showing the overexpression of gp130 subunit of IL-35 receptor in RA synovium while the expression of IL12Rb2 was comparable across different tissues. We hypothesize that the regulation of IL12Rb2 and gp130 by TNFa, showed in our in vitro experiments and their differential expression in cell types may contribute to unique effect of IL-35 in RA synovium. 5. Conclusion In conclusion, we demonstrated increased expression of both IL-35 (p35/EBI3) subunits in RA synovial tissue that were localized in several immune cells and synovial fibroblasts. We showed regulation of IL-35 receptor chains by a pro-inflammatory mediator and differential expression of its gp130 subunit in RA synovium. Consistent with previous reports, we confirmed that IL-35 subunits are upregulated by TNFa. Furthermore, we found that IL-35 induces synthesis of some pro-inflammatory cytokines in mononuclear cells, which support that IL-35 is pro-inflammatory in RA. Funding This work was supported by Grant Project IGA No. 12440-4, SVV Project No. 264 511 and Project of Ministerstvo Zdravotnictví ˇ eské Republiky for conceptual development of research organizaC tion 023728. Acknowledgements The authors acknowledge Dr. Jakub Sikora for valuable help with confocal microscopy, Dr. Daniel Housa and Katerˇina Vadinská for assistance with immunohistochemistry, and Anna Kozáková and Marie Krabcová for technical assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2015.01.019. References [1] Klareskog L, Catrina AI, Paget S. Rheumatoid arthritis. Lancet 2009;373: 659–72. [2] Smolen JS, Aletaha D. The assessment of disease activity in rheumatoid arthritis. Clin Exp Rheumatol 2010;28:S18–27. [3] Senolt L, Vencovsky J, Pavelka K, Ospelt C, Gay S. Prospective new biological therapies for rheumatoid arthritis. Autoimmun Rev 2009;9:102–7. [4] Collison LW, Vignali DA. Interleukin-35: odd one out or part of the family? Immunol Rev 2008;226:248–62.
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